Metalloenzymes are important in many critical life processes and an understanding of structure-function relationships in general is expected to contribute to health-related goals. X-ray absorption spectroscopic (XAS) investigations designed to elucidate the electronic and molecular structures of the active sites of a variety of metalloenzymes will be carried out. State-of-the-art collection and data analysis techniques will be used to extract detailed structural information. In each case, structure-function relationships will be developed by determining structural changes accompanying redox and/or substrate-binding steps of the enzyme's catalytic cycle. The ultimate goal is to understand the molecular mechanisms of catalysis by these enzymes. Studies on cytochrome c oxidases from beef heart and Paracoccus denitrificans (cytochromes aa3), and E. coli (cytochrome bo) are designed to elucidate the Cu-Fe binuclear O2-interaction site structure in the resting and activated states. Examples of all known types of nickel-containing enzymes, including jackbean urease, Klebsiella aerogenes urease, Desulfovibrio gigas [NiFe]hydrogenase, D. baculatus [NiFeSe]hydrogenase, Clostridium thermoaceticum CO dehydrogenase, and Methanobacterium thermoautotrophicum S-methyl coenzyme-M reductase, will be compared in terms of their nickel- site structures. A newly discovered nickel-binding protein from K. aerogenes (the ureE protein) that is apparently involved in urease metallocenter biosynthesis will be structurally characterized. Comparative studies are also planned on amine oxidases from a number of sources to elucidate the local structural environment of the cooper sites and their involvement with the organic topa cofactor in oxidation of primary amines. Other copper enzymes including galactose oxidase and N2O reductase will be examined as a general approach to understanding the varied roles of copper sites in metalloenzymes. XAS will also be used to examine Zn-containing matrix metalloproteinases (thought to be important in developmental process in normal growth and disease states, such as rheumatoid arthritis and cancer), recombinant rubredoxin variants, and metal-substituted [MFe3S4]ferredoxins from Pyrococcus furiosus.